Project Summary/Abstract: Electronic cigarette (e-cig) usage is on the rise, particularly among youths; however, their potential for harm is not understood, complicating development of informed regulatory strategies. The lack of data on e-cig related harm is, in part, due to the lack of specific biomarkers for exposure to e-cig aerosols. We found that e-cig aerosol contains highly reactive free radicals that can cause oxidative damage to the user. Free radicals can damage numerous cellular pathways possibly contributing to the progression of cancers and other diseases. Detection of e-cig free radicals can be accomplished by trapping with spin traps (e.g., DMPO) and analysis by electron paramagnetic resonance (EPR) spectroscopy. Preliminary research with EPR shows that free radicals produced in the e-cig aerosol by e-liquid solvents, propylene glycol (PG) and glycerin (GLY), common to all e-cigs, display unique structural characteristics. Our objective is to identify these free radical structures and utilize their unique structure to develop an e-cig specific biomarker of exposure. The specific aims of the proposed research are: (Aim 1) To determine the structures of the free radicals produced by PG and GLY in e-cigs; (Aim 2) To determine the primary targets of and adducts formed from free radical assault in the tissue of e-cig exposures in rodent models and possible metabolites formed from the these radical adducts in the serum of e-cig exposed rodent. This project represents an important research direction where a chemical/biological approach can inform tobacco regulatory science. As such, an important aspect of this application it to extend my background in areas relevant to translational science in addition to providing specific training in new biomarker-relevant research areas including metabolomics and free radical structural identification. To this end, my training will occur through a series of courses, relevant mentorship, and practical experience, each geared to ensure my transition to an independent researcher in the fields of biomarker development and regulatory science. Coursework, mentorship, conference participation, and practical training/experience will be completed during the K99 phase. During this phase, Aim 1 of the research plan will be completed and Aim 2 will be initiated (for completion during the R00 phase of the award). To accomplish the research aims, advanced pulsed EPR and mass spectroscopy approaches will be utilized for radicals produced by PG and GLY in e-cig aerosols. In a mouse exposure model, free radical exposure and targets of attack in the lung will be determined using a novel in vivo DMPO/anti-DMPO antibody approach. This will allow for the identification of specific protein and DNA adducts by TOF-MS. A post-DMPO exposure study will consist of an untargeted pairwise metabolomics approach to look at changes in metabolite profiles before and after e-cig exposure. By leveraging the unique structures of e-cig produced free radicals and their targets of attack in the lung, biomarkers of exposure specific to e-cig aerosols can be identified and used to develop regulatory strategies aimed at reducing harm from e-cig exposure.